Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4‐dioxane

Abstract

The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4-dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4-dioxane through chemical modification with poly(ethylene)-glycol (PEG, M(W) = 20 kDa) on these enzyme properties. In 1,4-dioxane a 10(4)-fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D(2)O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantioselectivity depended on the changes in protein structural dynamics within 1,4-dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media.

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

“…One strategy for broadening the range of activities over which enzyme catalysis and protein dynamics can be studied concurrently is to employ nonaqueous or organic solvents. For example, a correlation between molecular dynamics, as evidenced by a reduced rate of amide H/D exchange, and biocatalyst activity has been observed for PEGylated subtilisin Carlsberg (SC) in 1,4-dioxane (9).…”

Effects of Polyethylene Glycol on Bovine Intestine Alkaline Phosphatase Activity and Stability

“…Chiu et al reported that pegylation activates and stabilizes trypsin, although the mechanisms are not known. 39) In non-aqueous solution, pegylation increases the activities of various enzymes such as lipase 40) and subtilisin Carlsberg, 41) but in aqueous solution, it decreases the activities of various enzymes such as -chymotrypsin 42) and lysozyme. 43) In the case of -chymotrypsin, pegylation decreased the k cat values without changing the K m values.…”

UV-curable enzymatic antibacterial waterborne polyurethane coating

“…S2). Lysozyme has seven NH2 residues from lysozyme available for modification [30]. Thus, two (6 × 35%≈2.1) amine groups on enzyme molecule were modified.…”